Preparation method of single-cell nano coating, single-cell nano coating and application thereof

Abstract

The application discloses a preparation method of a single-cell nanometer coating, the single-cell nanometer coating and application thereof, and belongs to the technical field of biological medicines. The technical problems to be solved are to provide a simple, efficient and low-cost preparation method of the single-cell nanometer coating, and to improve the biocompatibility, long-term safety and uniformity of the single-cell nanometer coating. The technical solution points are a preparation method of a single-cell nanometer coating, which comprises the following steps: S1, adding a reagent containing a modified material into a cell suspension to obtain a mixed solution, wherein the modified material comprises a functionalized polyomithine derivative; and S2, incubating the mixed solution, so that the cells and the modified material react, and the modified material forms a single-cell nanometer coating on the surface of each cell.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and relates to cell surface modification and biopharmaceutical manufacturing, as well as the field of cell membrane engineering. Specifically, it relates to a method for preparing a single-cell nanocoating, the single-cell nanocoating, and its applications. Background Technology

[0002] For understanding the technical content of this invention:

[0003] Cell surface modification, as a key bioengineering technology, endows cells with new physical, chemical, or biological properties, and has broad application prospects in biomedical fields such as cell labeling and tracing, targeted drug delivery, cell therapy, and tissue engineering. The core objective of cell surface modification is to achieve precise regulation of cell function while maximally maintaining the cell's natural activity and function. Among these technologies, single-cell nanocoatings can construct continuous, controllable, and functionally specific interfacial layers on the surface of a single living cell, further endowing cells with new functions beyond their natural properties without significantly interfering with core cellular life activities.

[0004] Relevant patent documents retrieved: This document, published in China (CN112807289A) on May 18, 2021, discloses a method for modifying the surface of living cells based on nanoparticles and the nanoparticles used therein. The method includes the following steps: Step 1, preparing nanoparticles with -NH2, SH, or -COOH on their surface; Step 2, loading one or more bioactive substances onto the nanoparticles obtained in Step 1 to obtain nanoparticles loaded with one or more bioactive substances; Step 3, using active PEG (NHS-PEG-MAL, CHO-PE)... Step 2 involves modifying the surface of nanoparticles with G-MAL or EDC and NH2-PEG-MAL to obtain activated nanoparticles with maleimide (-MAL) or active ester (-NHS) groups. Step 4 involves collecting and culturing cells in the logarithmic growth phase, adding the activated nanoparticles from step 3 for incubation, and removing unreacted nanoparticles. Step 5 involves blocking the active groups on the surface of the nanoparticles linked to the cells with PEG-SH or PEG-NH2 to obtain surface-modified live cells carrying bioactive substances.

[0005] Relevant non-patent literature retrieved: Journal or book title: Life Sciences; Article title: Physicochemical Modification of Cell Surfaces; Volume No.: 2003, (02): 101-103; Publication date: 2003.04.15; This paper uses chemical materials such as polyethylene glycol, sodium alginate, chitosan, and polylysine to leverage the advantages of the intersection of chemistry, physics, and biology to carry out physicochemical reactions on the surface of graft cells, modifying and altering cell surface antigen molecules, which is expected to open up new avenues for overcoming transplant rejection.

[0006] The prior art represented by the aforementioned literature has at least the following unresolved technical problems or defects: (1) Biocompatibility and long-term safety issues: The biotoxicity, biodegradability, degradation product toxicity, and long-term in vivo immunogenicity of some synthetic polymers or inorganic nanomaterials used in existing methods have not been fully verified. Long-term in vivo application of some nanomaterials (such as silicon-based materials) may induce immune or inflammatory responses, and the degradation process may alter the pH of the local microenvironment, affecting normal cellular physiology. Especially during clinical translation, the safety of long-term in vivo application still needs further verification.

[0007] (2) Complex preparation and high cost: The preparation of most single-cell nanocoatings relies on precise layer-by-layer deposition or in-situ polymerization techniques, which usually involve cumbersome multi-step chemical reactions, are complex and time-consuming, and are difficult to control. At the same time, the high cost of some preparation materials (such as silicon-based materials) further limits their economic feasibility in actual clinical applications that require large-scale cell processing (such as large-scale cell therapy product production), becoming an important bottleneck for technology transfer.

[0008] (3) Lack of existing technical solutions: Existing technologies such as layer-by-layer self-assembly or in-situ polymerization usually result in uneven coating thickness (usually at the micrometer or submicrometer scale), poor uniformity of modification in cells, making it difficult to achieve a truly uniform single-cell coating, and single-cell coatings are difficult to achieve at the nanoscale.

[0009] Therefore, traditional cell surface modification methods suffer from problems such as complex operation, low modification efficiency, impaired cell viability, and poor coating uniformity and consistency. Relevant evidence includes: Patent document CN112807289A, which involves multiple steps such as nanoparticle preparation, loading, activation, incubation, and subsequent blocking. The repeated centrifugation, washing, and medium changes introduce significant mechanical stress, leading to irreversible loss of cell viability and function. Furthermore, the nanoparticles are silica with radial pores, which are costly and easily induce cellular immune or inflammatory responses. Non-patent literature, "Physicochemical Modification of Cell Surfaces," does not address single-cell nanocoatings, making it difficult to achieve uniform coating formation on single-cell surfaces and resulting in poor controllability.

[0010] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: In developing this invention, the inventors attempted to use hydrogels to three-dimensionally embed cells, hoping to form an "artificial extracellular matrix" through a polymer network to achieve immune isolation and functional modification. However, this approach has the following technical drawbacks: Drawback 1: Three-dimensional embedding leads to drastic changes in the cellular microenvironment: the hydrogel network density is 10-50 mg / mL, with a local viscosity >1000 cP, resulting in cells sensing a rigid matrix; Drawback 2: Material transport barrier: the hydrogel mesh size is 5-50 nm, creating diffusion resistance to nutrients / metabolites, leading to decreased cellular metabolic activity; Drawback 3: The contradiction between immune isolation and functional requirements: it is impossible to balance the dual needs of "protection" and "function"; Drawback 4: Cell damage from the cross-linking process: mTG catalyzes the production of NH3, raising the local pH to 8.5, causing cellular alkalosis; Drawback 5: Uncontrollable in vivo behavior: after gelatin enzymatic hydrolysis, cells disperse disorderly, lose targeting, and long-term retention triggers foreign body reaction (FBR).

[0011] In developing this invention, the inventors also attempted to use LBL (Layer-by-Layer) self-assembly technology to encapsulate cell surfaces with multiple layers of polyelectrolytes, hoping to construct a "nanocoating" of controllable thickness through electrostatic interactions to achieve surface functionalization and immune regulation. However, this approach has the following technical drawbacks: Drawback 1: Non-specific adsorption driven by electrostatic interactions: LBL relies on the electrostatic adsorption of polyelectrolytes on the cell surface (mainly negatively charged), resulting in low binding energy. The physiological environment (high ionic strength, serum protein competition) leads to rapid dissociation; Drawback 2: Spatial hindrance and inhibition of membrane protein function due to the multilayer structure: Each polyelectrolyte layer is 5-8 nm thick, and the total thickness after 5 layers is 25-40 nm. nm, forming a dense polymer barrier, physically burying membrane receptors, reducing ligand binding capacity, hindering cell signal transduction, reducing ERK phosphorylation levels, and inhibiting proliferation and migration functions; Defect 3, immune recognition and clearance induced by surface charge: Although the negatively charged terminal layer reduces complement activation, it promotes macrophage recognition, and regardless of positive or negative charge, the LBL coating significantly increases reticuloendothelial system clearance, shortening the in vivo half-life; Defect 4, cytotoxicity of polyelectrolytes: after primary amine protonation, membrane penetration occurs, mitochondrial depolarization leads to apoptosis, sulfonic acid groups interfere with membrane potential, cell metabolic activity decreases, and differences in deacetylation degree lead to uneven charge density, causing membrane damage in locally high-charge areas; Defect 5, cumulative damage and poor controllability of multilayer process: each washing and centrifugation step generates mechanical stress, multilayer accumulation leads to decreased cell viability, interlayer interactions are sensitive to temperature, pH, and ionic strength, and there are large batch-to-batch thickness differences; functional molecules are doped into the polycation layer, and release is controlled by multilayer diffusion, exhibiting burst release rather than sustained release.

[0012] The hydrogel three-dimensional embedding scheme has systemic defects in terms of material transport, cell function preservation, in vivo behavior controllability and process safety, while the LBL layer-by-layer self-assembly scheme has systemic defects in terms of coating stability, immune safety, membrane protein function preservation and process controllability. Both also have the fundamental limitation of "physical interaction". Summary of the Invention

[0013] The purpose of this invention is to provide: This invention discloses a method for preparing a single-cell nanocoating, the single-cell nanocoating itself, its applications, and related technologies, aiming to solve the technical problems, or combinations thereof, of existing cell surface modification methods, such as complex operation, low modification efficiency, impaired cell activity, and poor coating uniformity and consistency. This invention utilizes functionalized polysarcosine derivative covalent anchoring technology, replacing electrostatic interactions with chemical bonds, multilayers with single layers, and high charges with electrically neutral bonds, fundamentally solving the aforementioned problems. This represents a paradigm shift in cell membrane engineering from physical coating to chemical anchoring.

[0014] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0015] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0016] Definitions of standard terms can be found in the references “Cell Biology (5th Edition), Higher Education Press, authors: Ding Mingxiao, Wang Xizhong, Zhang Chuanmao, Chen Jianguo, et al., 2020-05” and “Modern Molecular Biology (5th Edition), Higher Education Press, authors: Zhu Yuxian, Li Yi, Zheng Xiaofeng, and Guo Hongwei, 2019-06-19”.

[0017] Unless otherwise stated, conventional methods within the scope of the art, such as cell washing, cell centrifugation, and cell resuspension, shall be used.

[0018] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0019] The term "DSPE-PSar-NHS" used in this article refers to: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(sarcosine)]-N-hydroxysuccinimide ester, which is translated into Chinese as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polysarcosine]-N-hydroxysuccinimide ester.

[0020] The term “DSPE-PSar-Maleimide” used in this article refers to: 1,2-distearoyl-sn-glycero-3-hosphoethanolamine-N-[poly(sarcosine)]-N-maleimide, which is translated into Chinese as 1,2-distearoyl-sn-glycero-3-hosphoethanolamine-N-[polysarcosine]-N-maleimide.

[0021] The term “DSPE-PSar-Biotin” used in this article refers to: 1,2-distearoyl-sn-glycero-3-hosphoethanolamine-N-[poly(sarcosine)]-N-biotin, which is translated into Chinese as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[polysarcosine]-N-biotin.

[0022] The term "SOPE-PSar2000-NHS" used in this article refers to: 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine-Poly(N-methylglycine)(2000)-N-Hydroxysuccinimide Ester, which is translated into Chinese as monostearate-monooleoyl-phosphatidylethanolamine-polysarcosine(2000)-N-hydroxysuccinimide ester.

[0023] The term “Cholesterol-PSar2000-NHS” used in this article refers to Cholest-5-en-3β-yl-Poly(N-methylglycine)(2000)-N-Hydroxysuccinimide Ester, which is translated into Chinese as cholesterol-polysaccharide(2000)-N-hydroxysuccinimide ester.

[0024] The term “biocompatibility” used in this article refers to the property of a living organism’s tissues to respond to inactive materials. It involves the interaction between the material and the host, and is manifested as a dynamic interaction process between the material and the organism, including both biological reactions and changes in the physicochemical properties of the material.

[0025] As used in this article, the term "nanocoating" refers to a continuous thin layer or film structure with a thickness at the nanoscale (typically 1-1000 nanometers) constructed on the surface of a material or biological interface by physical, chemical, or biological methods.

[0026] In a first aspect, the present invention provides: a method for preparing a single-cell nanocoating, the method comprising: S1. Add a reagent containing a modifying material to a cell suspension to obtain a mixture, wherein the modifying material includes a functionalized polysarcosine derivative; S2. Incubate the mixture to allow the cells and the modified material to react and form the single-cell nanocoating.

[0027] Specifically, in step S2, the mixture is incubated to allow the cells and the modifying material to react, enabling the modifying material to form the single-cell nanocoating on the surface of each cell.

[0028] Furthermore, the structure of the functionalized polysarcosine derivative includes a front-end-intermediate polymer, wherein the front end is independently selected from DSPE (distearylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), DMPE (dimyristoylphosphatidylethanolamine), DPPE (dispalmitoylphosphatidylethanolamine), DEPE (ditransoleoylphosphatidylethanolamine), SOPE (stearoylphosphatidylethanolamine), DSG (distearylglycerol), Dialkylglycerol, POPE (palmitoylphosphatidylethanolamine), Cholesterol, Dioctadecyl, Tocopherol, or Stearyl, and the intermediate polymer includes PSar.

[0029] Furthermore, the functionalized polysarcosine derivative also includes a back-end activating group. The structure of the functionalized polysarcosine derivative is a front-end-intermediate polymer-back-end activating group, wherein the back-end activating group is independently selected from NHS ester (N-hydroxysuccinimide ester), Sulfo-NHS ester (sulfonyl-NHS ester), NHS carbonate (NHS carbonate), PFP ester (pentafluorophenol ester), DBCO (dibenzocyclooctylene), Tetrazine (tetraazine), Norbornene (norbornene), Cyclooctyne (cyclooctylene), TCO (trans-cyclooctylene), Azide (azide), Maleimide (maleimide), Thiol-ene (thiol-ene), Vinyl sulfone (ethylene sulfone), Pyridyl disulfide (pyridyl disulfide), or Biotin (biotin).

[0030] For example, the functionalized polysarcosine derivative can be selected from DSPE-PSar-NHS ester, DSPE-PSar-Sulfo-NHS ester, DSPE-PSar-NHS carbonate, DSPE-PSar-PFP ester, DSPE-PSar-DBCO, DOPE-PSar-NHS ester, DOPE-PSar-Sulfo-NHS ester, DOPE-PSar-NHS carbonate, DOPE-PSar-PFP ester, DOPE-PSar-DBCO, DMPE-PSar-NHS ester, DMPE-PSar-Sulfo-NHS ester, DMPE-PSar-NHS carbonate, DMPE-PSar-PFP ester, DMPE-PSar-DBCO, DPPE-PSar-NHSester, DPPE-PSar-Sulfo-NHS ester, DPPE-PSar-NHS carbonate, DPPE-PSar-PFP ester, DPPE-PSar-DBCO, DEPE-PSar-NHS ester, DEPE-PSar-Sulfo-NHS ester, DEPE-PSar-NHScarbonate, DEPE-PSar-PFP ester, DEPE-PSar-DBCO, SOPE-PSar-NHS ester, SOPE-PSar-Sulfo-NHS ester, SOPE-PSar-NHS carbonate, SOPE-PSar-PFP ester, SOPE-PSar-DBCO, DSG-PSar-NHS ester, DSG-PSar-Sulfo-NHS ester, DSG-PSar-NHS carbonate, DSG-PSar-PFP ester, DSG-PSar-DBCO, Dialkylglycerol-PSar-NHS ester, Dialkylglycerol-PSar-Sulfo-NHS ester, Dialkylglycerol-PSar-NHS carbonate, Dialkylglycerol-PSar-PFP ester, Dialkylglycerol-PSar-DBCO, POPE-PSar-NHS ester, POPE-PSar-Sulfo-NHS ester, POPE-PSar-NHS carbonate, POPE-PSar-PFPester, POPE-PSar-DBCO, Cholesterol-PSar-NHS ester, Cholesterol-PSar-Sulfo-NHS ester, Cholesterol-PSar-NHScarbonate, Cholesterol-PSar-PFP ester, Cholesterol-PSar-DBCO, Dioctadecyl-PSar-NHS ester, Dioctadecyl-PSar-Sulfo-NHS ester, Dioctadecyl-PSar-NHS carbonate, Dioctadecyl-PSar-PFP ester, Dioctadecyl-PSar-DBCO, Tocopherol-PSar-NHS ester, Tocopherol-PSar-Sulfo-NHS ester, Tocopherol-PSar-NHS carbonate, Tocopherol-PSar-PFP ester、Tocopherol-PSar-DBCO、DSPE-PSar-Tetrazine、DSPE-PSar-Norbornene、DSPE-PSar-Cyclooctyne、DSPE-PSar-TCO、DSPE-PSar-Azide、DSPE-PSar-Maleimide、DSPE-PSar-Thiol-ene、DSPE-PSar-Vinyl sulfone、DSPE-PSar-Pyridyl disulfide、DSPE-PSar、DOPE-PSar-Tetrazine、DOPE-PSar-Norbornene、DOPE-PSar-Cyclooctyne、DOPE-PSar-TCO、DOPE-PSar-Azide、DOPE-PSar-Maleimide、DOPE-PSar-Thiol-ene、DOPE-PSar-Vinylsulfone、DOPE-PSar-Pyridyl disulfide、DOPE-PSar、DMPE-PSar-Tetrazine、DMPE-PSar-Norbornene、DMPE-PSar-Cyclooctyne、DMPE-PSar-TCO、DMPE-PSar-Azide、DMPE-PSar-Maleimide、DMPE-PSar-Thiol-ene、DMPE-PSar-Vinylsulfone, DMPE-PSar-Pyridyldisulfide, DMPE-PSar, DPPE-PSar-Tetrazine, DPPE-PSar-Norbornene, DPPE-PSar-Cyclooctyne, DPPE-PSar-TCO, DPPE-PSar-Azide, DPPE-PSar-Maleimide, DPPE-PSar-Thiol-ene, DPPE-PSar-Vinyl sulfone, DPPE-PSar-Pyridyl disulfide, DPPE-PSar, DEPE-PSar-Tetrazine, DEPE-PSar-Norbornene, DEPE-PSar-Cyclooctyne, DEPE-PSar-TCO, DEPE-PSar-Azide, DEPE-PSar-Maleimide, DEPE-PSar-Thiol-ene, DEPE-PSar-Vinyl sulfone, DEPE-PSar-Pyridyl disulfide、DEPE-PSar、SOPE-PSar-Tetrazine、SOPE-PSar-Norbornene、SOPE-PSar-Cyclooctyne、SOPE-PSar-TCO、SOPE-PSar-Azide、SOPE-PSar-Maleimide、SOPE-PSar-Thiol-ene、SOPE-PSar-Vinyl sulfones、SOPE-PSar-Pyridyldisulfide、SOPE-PSar、DSG-PSar-Tetrazine、DSG-PSar-Norbornene、DSG-PSar-Cyclooctyne、DSG-PSar-TCO、DSG-PSar-Azide、DSG-PSar-Maleimide、DSG-PSar-Thiol-ene、DSG-PSar-Vinyl sulfone、DSG-PSar-Pyridyldisulfide, DSG-PSar, Dialkylglycerol-PSar-Tetrazine, Dialkylglycerol-PSar-Norbornene, Dialkylglycerol-PSar-Cyclooctyne, Dialkylglycerol-PSar-TCO, Dialkylglycerol-PSar-Azide, Dialkylglycerol-PSar-Maleimide, Dialkylglycerol-PSar-Thiol-ene, Dialkylglycerol-PSar-Vinyl sulfone, Dialkylglycerol-PSar-Pyridyl disulfide, Dialkylglycerol-PSar, POPE-PSar-Tetrazine, POPE-PSar-Norbornene, POPE-PSar-Cyclooctyne, POPE-PSar-TCO, POPE-PSar-Azide, POPE-PSar-Maleimide, POPE-PSar-Thiol-ene, POPE-PSar-Vinyl sulfone, POPE-PSar-Pyridyl disulfide, POPE-PSar, Cholesterol-PSar-Tetrazine, Cholesterol-PSar-Norbornene, Cholesterol-PSar-Cyclooctyne, Cholesterol-PSar-TCO, Cholesterol-PSar-Azide, Cholesterol-PSar-Maleimide, Cholesterol-PSar-Thiol-ene, Cholesterol-PSar-Vinyl sulfone, Cholesterol-PSar-Pyridyl disulfide, Cholesterol-PSar, Dioctadecyl-PSar-Tetrazine, Dioctadecyl-PSar-Norbornene, Dioctadecyl-PSar-Cyclooctyne, Dioctadecyl-PSar-TCO, Dioctadecyl-PSar-Azide, Dioctadecyl-PSar-Maleimide, Dioctadecyl-PSar-Thiol-ene, Dioctadecyl-PSar-Vinylsulfone, Dioctadecyl-PSar-Pyridyl disulfide, Dioctadecyl-PSar, Tocopherol-PSar-Tetrazine, Tocopherol-PSar-Norbornene, Tocopherol-PSar-Cyclooctyne, Tocopherol-PSar-TCO, Tocopherol-PSar-Azide, Tocopherol-PSar-Maleimide, Tocopherol-PSar-Thiol-ene, Tocopherol-PSar-Vinyl sulfone, Tocopherol-PSar-Pyridyl disulfide, Tocopherol-PSar, Stearyl-PSar-NHS ester, Stearyl-PSar-Sulfo-NHS ester, Stearyl-PSar-NHS carbonate, Stearyl-PSar-PFP ester、Stearyl-PSar-DBCO、Stearyl-PSar-Tetrazine、Stearyl-PSar-Norbornene、Stearyl-PSar-Cyclooctyne、Stearyl-PSar-TCO、Stearyl-PSar-Azide、Stearyl-PSar-Maleimide、Stearyl-PSar-Thiol-ene、Stearyl-PSar-Vinyl sulfone、Stearyl-PSar-Pyridyl disulfide、Stearyl-PSar、DSPE-PSar-Biotin、 At least one of DOPE-PSar-Biotin, DMPE-PSar-Biotin, DPPE-PSar-Biotin, DEPE-PSar-Biotin, DEPE-PSar-Biotin, SOPE-PSar-Biotin, DSG-PSar-Biotin, Dialkylglycerol-PSar-Biotin, POPE-PSar-Biotin, Cholesterol-PSar-Biotin, Dioctadecyl-PSar-Biotin, Tocopherol-PSar-Biotin, Stearyl-PSar-Biotin, etc.

[0031] Furthermore, the molecular weight of PSa in functionalized polysarcosine derivatives can be 500 Da to 5000 Da.

[0032] Furthermore, the molecular weight of PSA in functionalized polysarcosine derivatives can be 2000 Da-5000 Da.

[0033] According to some embodiments of the present invention, the molecular weight of PSar in the functionalized polysarcosine derivative can, for example, be 500 Da, 1000 Da, 2000 Da, 3000 Da, 4000 Da, 5000 Da, or any value within the range of any two of the above values. The molecular weight of PSar affects the chain length of the functionalized polysarcosine derivative. Longer PSar chains (e.g., molecular weight 4000 Da-5000 Da) can provide better steric hindrance, reducing protein adsorption and immune responses, while shorter PSar chains (e.g., molecular weight 500 Da-1000 Da) can increase the reaction rate and shorten the modification time, making them suitable for scenarios requiring rapid modification.

[0034] Preferably, the functionalized polysarcosine derivative may be selected from at least one of DSPE-PSar2000-NHS, DSPE-PSar1000-NHS, DSPE-PSar5000-NHS, DSPE-PSar2000-Maleimide, DSPE-PSar2000-Biotin, SOPE-PSar2000-NHS, Cholesterol-PSar2000-NHS, etc.

[0035] For example, the functionalized polysarcosine derivative can be DSPE-PSar2000-NHS. DSPE-PSar2000-NHS has stable amide bonds, and the NHS (N-hydroxysuccinimide) activating group can specifically react with amino groups on the cell surface to form stable amide bonds. These chemical bonds are very stable under physiological conditions and are not easily hydrolyzed or broken. Furthermore, the flexibility and hydrophilicity of the PSar chain can buffer external physical and chemical shocks, further enhancing the stability of the coating. This comprehensively improves the chemical stability and durability of the single-cell nanocoating, enabling it to remain intact in complex physiological environments and preventing coating detachment or degradation.

[0036] For example, the functionalized polysarcosine derivative can be selected from at least one of DSPE-PSar2000-Biotin, DSPE-PSar1000-Biotin, DSPE-PSar5000-Biotin, etc., wherein biotin has extremely high affinity and can stably and specifically bind to streptavidin. Through the binding of biotin to streptavidin, multiple biomolecules can be linked to achieve multifunctional applications.

[0037] According to some embodiments of the present invention, in step S1, the concentration of the modifying material in the mixture can be between 10 µg / mL and 200 µg / mL, which is beneficial for better balancing high modification efficiency, high uniformity of the single-cell nanocoating on the cell surface, and good cell activity. This concentration can exemplary be 10 µg / mL, 30 µg / mL, 40 µg / mL, 50 µg / mL, 60 µg / mL, 70 µg / mL, 80 µg / mL, 90 µg / mL, 100 µg / mL, 150 µg / mL, 200 µg / mL, or any value within the range of any two of the above values. The concentration can further be between 40 µg / mL and 80 µg / mL, and even further between 45 µg / mL and 55 µg / mL, with 50 µg / mL being the preferred concentration.

[0038] According to some embodiments of the present invention, the concentration of the modifying material in the reagent containing the modifying material can be between 5 mg / mL and 20 mg / mL, which is beneficial for better balancing high modification efficiency, high uniformity of the single-cell nanocoating on the cell surface, and good cell activity. This concentration can exemplary be 5 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 18 mg / mL, 20 mg / mL, or any value within the range of any two of the above values. The concentration can further be between 8 mg / mL and 15 mg / mL, and preferably 10 mg / mL.

[0039] According to some embodiments of the present invention, the incubation temperature can be 0℃-37℃, which is beneficial to further improve the modification efficiency while maintaining good cell viability and inhibiting the endocytosis of the modified material by cells. The incubation temperature can exemplary be 0℃, 5℃, 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, or any value within the range of any two of the above values. The incubation temperature can further be 0℃-8℃, further can be 20℃-25℃, and further can be 30℃-37℃. The incubation temperature can still be further refined to 2℃-6℃, and preferably 4℃.

[0040] According to some embodiments of the present invention, the incubation time can be 10 min to 60 min, which is beneficial for sufficient modification reaction, further improving the uniformity of the single-cell nanocoating, while maintaining cell viability. The incubation time can exemplary be 10 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or any value within the range of any two of the above values. The incubation time can further be 20 min to 40 min, and preferably 30 min.

[0041] According to some embodiments of the present invention, the cells may be selected from at least one of the following: mesenchymal stem cells, natural killer cells (including surface-modified natural killer cells, such as chimeric antigen receptor natural killer cells), T cells (including surface-modified T cells, such as chimeric antigen receptor T cells), pancreatic islet cells, kidney cells, cardiomyocytes, liver cells, erythrocytes, various tumor cell lines, primary fibroblasts, chondrocytes, neurons, neural stem cells, dopaminergic precursor cells, pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells, etc., whose cell membrane surface contains amino groups.

[0042] According to some embodiments of the present invention, a method for preparing a cell suspension includes: washing the cells and adjusting the cell concentration to 5 × 10⁻⁶ using a buffer solution. 5 cells / 300µL - 5×10 6 Cells / 300µL were used to obtain a cell suspension. Washing the cells helps remove impurities and residual culture medium components from the cell surface, ensuring the accuracy of subsequent reactions. Adding buffer to adjust the cell concentration helps to achieve an appropriate cell concentration in the reaction system, further improving the uniformity of modification of the modified material. An exemplary cell concentration could be 5 × 10⁻⁶ cells / 300µL. 5 cells / 300µL, 1×10 6 cells / 300µL, 2×10 6 cells / 300µL, 3×10 6 cells / 300µL, 4×10 6 cells / 300µL, 5×10 6 cells / 300µL or any value within the range of any two of the above values, preferably 1×10 6 cells / 300µL.

[0043] Pre-cooling the cells during the preparation of cell suspensions can help reduce the rate of endocytosis and decrease the endocytosis of the modification material by the cells during the modification reaction.

[0044] According to some embodiments of the present invention, the buffer solution may include at least one of HEPES buffer, Tris buffer, and PBS buffer, which is beneficial for maintaining the pH stability of the reaction system. Furthermore, ethylenediaminetetraacetic acid (EDTA) may be added to the buffer solution to reduce metal ion interference. HEPES buffer can stabilize the pH of the reaction system; Tris buffer has better buffering capacity in the neutral pH range and is suitable for various biochemical reactions and reaction conditions requiring higher buffering capacity; PBS buffer (phosphate buffer) has a simple composition, low cost, and good biocompatibility and stability.

[0045] According to some embodiments of the present invention, after step S2, the preparation method may further include: centrifuging and washing the cells in the mixture to remove unreacted modification materials. Specifically, the cells can be washed with PBS or other cell washing solutions to completely remove free modification materials, which is beneficial for further removing unbound modification materials and improving the purity of the modification effect.

[0046] Furthermore, prior to the step of centrifuging and washing the cells in the mixture, the preparation method may further include: adding a terminator to the mixture to terminate the reaction. The terminator binds to the modified material, terminating the reaction between the modified material and the cells. Specifically, the terminator may contain at least one of glycine (Gly), ethanolamine, and hydroxylamine. Glycine balances a relatively fast reaction rate with milder reaction conditions; ethanolamine further improves the efficiency of termination and maintains cell viability; hydroxylamine offers milder reaction conditions and has less impact on cell viability.

[0047] According to some embodiments of the present invention, the thickness of the single-cell nanocoating can be 1nm-10nm, and can be, for example, 1nm, 3nm, 5nm, 7nm, 9nm, 10nm, or any value within the range of any two of the above values, further can be 3nm-7nm, and even further can be 5nm-6nm.

[0048] Secondly, the present invention provides a single-cell nanocoating, which is prepared by the above-described method for preparing single-cell nanocoatings.

[0049] Thirdly, the present invention provides: a surface-engineered cell having the above-mentioned single-cell nanocoating.

[0050] Furthermore, the surface-engineered cells are living cells.

[0051] Furthermore, each surface-engineered cell has the aforementioned single-cell nanocoating.

[0052] Fourthly, the present invention provides a pharmaceutical composition comprising the aforementioned surface-engineered cells. The surface-engineered cells in this pharmaceutical composition can carry multiple drugs or biomolecules, achieving sustained and precise drug release, significantly improving drug loading and therapeutic efficacy. This is because the single-cell nanocoating on the surface of the surface-engineered cells contains polysarcosine (PSar) chains, which can be chemically linked to multiple drug molecules, biomarkers, or fluorescent probes. This modifiability allows functionalized polysarcosine derivatives to carry multiple functional molecules, significantly increasing drug loading. Furthermore, the balance between the hydrophobicity and hydrophilicity of the PSar chains can regulate the drug release rate, achieving a sustained-release effect. By further adjusting the length and modification density of the PSar chains, the drug release rate can be controlled more precisely.

[0053] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0054] Furthermore, the pharmaceutical composition can be formulated into various dosage forms, including but not limited to: injections, implants, or gels or sprays for topical application.

[0055] Fifthly, the present invention provides: an engineered cell preparation comprising the above-described surface-engineered cells.

[0056] Furthermore, the engineered cell preparation can be a cryopreservation preparation or a cell suspension, etc.

[0057] In a sixth aspect, the present invention provides a kit comprising the aforementioned surface-engineered cells.

[0058] Furthermore, the kit may also include at least one of a fluorescent marker, a diluent, a syringe, etc.

[0059] In a seventh aspect, the present invention provides the use of the above-described single-cell nanocoating or the above-described surface-engineered cells or the above-described pharmaceutical composition or the above-described engineered cell preparation in cell labeling, drug delivery and preparation of medicaments for cell therapy.

[0060] Single-cell nanocoatings or surface-engineered cells not only enable cell surface modification but also facilitate various applications such as drug delivery, cell labeling, and biosensors, demonstrating broad versatility. This is due to the multifunctionality of the PSA chains in single-cell nanocoatings. These chains can chemically link various bioactive molecules, such as drugs, fluorescent probes, or biomarkers, enabling functionalized polysarcosine derivatives for diverse biomedical applications. Furthermore, the posterior activating groups (such as NHS, Maleimide, and biotin) are versatile, capable of binding to a variety of biomolecules and providing extensive modification capabilities. Particularly in the preparation of drugs for cell therapy, single-cell nanocoatings can reduce blood flow shear stress after cells enter the bloodstream and protect cells from phagocytosis (immune responses) caused by cell damage as they pass through the pulmonary capillary brush. These single-cell nanocoatings employ a dual physical-chemical shield, blocking opsonin adsorption and macrophage pseudopodia contact to inhibit phagocytosis of intact cells.

[0061] For example, in cell labeling applications, PSA chains in single-cell nanocoatings can be linked to fluorescent probes to serve as non-invasive, long-lasting cell tracers.

[0062] For example, in drug delivery applications, PSA chains in single-cell nanocoatings can be linked to drugs to achieve targeted drug delivery.

[0063] For example, in the preparation of drugs for cell therapy, the single-cell nanocoating can reduce blood flow shear stress after cells enter the bloodstream and protect cells from phagocytosis (immune response) caused by cell damage as they pass through the pulmonary capillary brush. The single-cell nanocoating takes on a dual physical-chemical shield, blocking opsonin adsorption and macrophage pseudopodia contact to inhibit phagocytosis of intact cells.

[0064] Examples 1-20 of this invention at least support the protection scope of "method for preparing single-cell nanocoatings".

[0065] The term "preparation method of single-cell nanocoating" is summarized from the foregoing explanation and / or the corresponding preparation methods in Examples 1-20. Therefore, those skilled in the art can reasonably infer that "preparation method of single-cell nanocoating," its subordinate concepts, its essentially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope of "preparation method of single-cell nanocoating." Replacing "preparation method of single-cell nanocoating" with "method for preparing single-cell nanocoating," etc., still falls within the protection scope of this invention.

[0066] Examples 1-20 of this invention at least support the protection range of the "single-cell nanocoating".

[0067] The term "single-cell nanocoating" is derived from the foregoing explanation and / or the corresponding single-cell nanocoatings in Examples 1-20. Therefore, those skilled in the art can reasonably infer that "single-cell nanocoating," its subordinate concepts, its substantially equivalent technical means, and technical means that can replace it within the scope of conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of "single-cell nanocoating." Replacing "single-cell nanocoating" with "single-cell nanointerface layer" or "single-cell nanomodification layer," etc., still falls within the protection scope of this invention.

[0068] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention provides a method for preparing single-cell nanocoatings, which has better technical effects, specifically reflected in the following aspects: 1. The modified materials employed possess low cost, high biocompatibility, low immunogenicity, and long-term safety: The modified materials include functionalized polysarcosine derivatives. These derivatives (e.g., DSPE-PSar-NHS) are cell membrane analogs that can fuse well with cell membranes, reducing cytotoxicity at the source. PSar is a long-proven biocompatible material; its modification effectively reduces non-specific adsorption, significantly lowers immunogenicity, and avoids triggering immune or inflammatory responses. Furthermore, the entire modification system is based on widely validated PSarization technology, a gentle modification process that does not produce acidic byproducts or other harmful substances, ensuring safety for long-term in vivo application. In addition, functionalized polysarcosine derivatives are common biomaterials with ample market supply and relatively low prices. Compared to silicon-based materials or complex polymer nanoparticles, they are more cost-effective and suitable for large-scale applications.

[0069] 2. The single-cell nanocoating exhibits excellent stability and durability: The single-cell nanocoating prepared by the method provided in this invention possesses extremely high chemical stability and durability, maintaining its integrity even in complex physiological environments and preventing coating detachment or degradation. This is attributed to the protective effect of the PSA chains; the flexibility and hydrophilicity of the PSA chains buffer external physical and chemical shocks, effectively enhancing the coating's stability. Furthermore, when the single-cell nanocoating contains activating groups such as NHS (N-hydroxysuccinimide), it can specifically react with amino groups on the cell surface to form stable amide bonds. These chemical bonds are highly stable under physiological conditions and are not easily hydrolyzed or broken, further improving the stability and durability of the single-cell nanocoating.

[0070] 3. The preparation process is simple, efficient, precise, controllable, and low-cost: A nanoscale coating can be inserted into the cell surface simply by directly incubating the modifying material with cells using a simple shaking device. The process involves few steps, is concise, and is easy to standardize. This invention can also efficiently modify cell surfaces, with a gentle modification process that has minimal impact on cell viability and high modification efficiency. Furthermore, the thickness and uniformity of the single-cell nanocoating can be more precisely adjusted by controlling the concentration of the functionalized polysarcosine derivative, reaction time, and temperature. The preparation method of this invention can replace existing hydrogel encapsulation methods, reducing costs by at least 50% and further improving cell encapsulation efficiency.

[0071] 4. Wide applicability and practicality: The PSar chain of functionalized polysarcosine derivatives can serve as a universal linker, conveniently coupling various functional molecules such as drug molecules, fluorescent probes, and targeting ligands (e.g., antibodies, peptides) via chemical bonds, achieving "one material for multiple uses." This is beneficial for the preparation method of the single-cell nanocoating of this invention and for the large-scale promotion and application of single-cell nanocoatings. The modifiability of the PSar chain effectively increases the drug loading capacity of the single-cell nanocoating. Simultaneously, the balance between hydrophobicity and hydrophilicity of the PSar chain can regulate the drug release rate, achieving a sustained-release effect and exhibiting good refill capacity and drug release control. By further adjusting the length and modification density of the PSar chain, the drug release rate can be controlled more precisely.

[0072] In summary, the method for preparing single-cell nanocoatings provided by this invention has advantages such as simple and efficient process, precise controllability and low cost. The prepared single-cell nanocoatings have advantages such as high biocompatibility, low immunogenicity and long-term safety. They can efficiently, stably and gently modify the cell surface, and have important research value and application prospects. Attached Figure Description

[0073] Figure 1 This is a schematic flowchart of the method for preparing single-cell nanocoatings in Example 1 of the present invention.

[0074] Figure 2 These are images of modified cells observed under a microscope in Examples 1-4 of this invention.

[0075] Figure 3 These are images of modified cells observed under a microscope in Examples 5-8 of this invention.

[0076] Figure 4 These are images of modified cell states observed under a microscope in Examples 9-12 of this invention.

[0077] Figure 5 These are images of modified cell states observed under a microscope in Examples 13-16 of this invention.

[0078] Figure 6These are images of modified cell states observed under a microscope in Examples 17-20 of this invention.

[0079] Figure 7 This is a microscopic observation of the modified cell state in Comparative Example 1 of the present invention.

[0080] Figure 8 This is a microscopic observation of the modified cell state in Comparative Example 2 of the present invention. Detailed Implementation

[0081] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0082] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0083] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0084] The main reagents and instruments used in the following examples are: DSPE-PSar2000-NHS (Ruixin Biotechnology Co., Ltd., R-d6-0251-2K), DSPE-PSar1000-NHS (Ruixin Biotechnology Co., Ltd., R-d6-0251-1K), DSPE-PSar5000-NHS (Ruixin Biotechnology Co., Ltd., R-d6-0251-5K), DSPE-PSar2000-Maleimide (Ruixin Biotechnology Co., Ltd., R-d6-0253-2k), DSPE-PSar2000-Biotin (Ruixin Biotechnology Co., Ltd., R-d6-0261-2k), Cholesterol-PSar2000-PFP (Ruixin Biotechnology Co., Ltd., custom-made, no catalog number), DSPC-PSar2000-NHS (Ruixin Biotechnology Co., Ltd., R-d6-0251-2k), Cholesterol-PSar2000-PFP (Ruixin Biotechnology Co., Ltd., custom-made, no catalog number), DSPC-PSar2000-NHS (Ruixin Biotechnology Co., Ltd., R-d6-0251-2K), and DSPE-PSar2000-NHS (Ruixin Biotechnology Co., Ltd., R-d6-0251-2K). Biotechnology company (customized, no catalog number), DSG-PSar2000-NHS (Ruixin Biotechnology Company, customized, no catalog number), Dioctadecyl-PSar2000-DBCO (Ruixin Biotechnology Company, customized, no catalog number), Tocopherol-PSar2000-Thiol-ene (Ruixin Biotechnology Company, customized, no catalog number), DSPC-PSar2000 (Ruixin Biotechnology Company, customized, no catalog number), mesenchymal stem cells (MSCs, Beijing Weilan Times Biotechnology Co., Ltd.), PBS solution (ThermoFisher, catalog number 10010023), DPBS solution (ThermoFisher, catalog number 14190144), fluorescence microscope (Mingmei Company, catalog number MI40), laser scanning confocal microscope (Zeiss Company, catalog number LSM 880), HEPES buffer (ThermoFisher, catalog number 15630130), Tris buffer (ThermoFisher, catalog number AM9850G).

[0085] Example 1 Raw material preparation: (1) Cell suspension: Adipose-derived mesenchymal stem cells (MSCs) were washed twice with DPBS solution for about 5 min each time, and the cells were pre-cooled at 4°C (the pre-cooling temperature is the same as the incubation temperature in the subsequent preparation method) for 10 min. After pre-cooling, the cells were centrifuged at 4°C, the supernatant was discarded, and the cell concentration was adjusted to 1×10⁻⁶ cells / mL with pre-cooled buffer. 6 cells / 300µL. The buffer solution consisted of 81.5mL physiological saline, 4mL 0.5M HEPES buffer at pH 8.0, 0.1mL 0.5M EDTA, and 14.4mL water for injection.

[0086] (2) Reagent containing the modified material: Weigh DSPE-PSar2000-NHS, dissolve it with an appropriate amount of DMSO, and prepare a reagent containing the modified material with a concentration of 10 mg / mL.

[0087] The structural formula of DSPE-PSar2000-NHS is: .

[0088] Preparation method (steps S1 and S2 for forming the single-cell nanocoating can be found in the documentation) Figure 1 (as shown) Step 1: Add 1.5 µL of reagent containing the modification material to 300 µL of cell suspension to obtain a mixture. The modification material is DSPE-PSar2000-NHS, and the concentration of DSPE-PSar2000-NHS in the mixture is 50 µg / mL (approximately 22 µmol / L).

[0089] Step 2: Incubate the mixture at 4°C for 30 minutes to allow the cells and the modification material to react, so that the modification material forms a single-cell nanocoating on the surface of each cell.

[0090] Step 3: Add 1 mL of terminator (100 mM Gly-PBS) to the incubated mixture and react at room temperature for 5 min to terminate the reaction between the cells and the modified material. After termination, wash the cells twice with 10 mL of PBS solution for about 5 min each time to remove unreacted modified material.

[0091] Example 2: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is: DSPE-PSar2000-NHS is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 5 mg / mL. That is, in step 1, the concentration of DSPE-PSar2000-NHS in the reagent containing the modifying material is 5 mg / mL. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0092] Example 3: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is: weighing DSPE-PSar2000-NHS, dissolving it in an appropriate amount of DMSO, and preparing a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the concentration of DSPE-PSar2000-NHS in the reagent containing the modifying material is 20 mg / mL. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0093] Example 4: The only difference from Example 1 is that in step 1, the concentration of DSPE-PSar2000-NHS in the mixture is 20 µg / mL. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0094] Example 5: The only difference from Example 1 is that in step 1, the concentration of DSPE-PSar2000-NHS in the mixture is 100 µg / mL. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0095] Example 6: The only difference from Example 1 is that the pre-cooling temperature in raw material preparation and the incubation temperature in step 2 are both 20°C. The preparation methods for the remaining single-cell nanocoatings are basically the same as in Example 1.

[0096] Example 7: The only difference from Example 1 is that the pre-cooling temperature in raw material preparation and the incubation temperature in step 2 are both 37°C. The preparation methods for the remaining single-cell nanocoatings are basically the same as in Example 1.

[0097] Example 8: The only difference from Example 1 is that in step 2, the incubation time is 15 minutes. The rest of the preparation method for the single-cell nanocoating is basically the same as in Example 1.

[0098] Example 9: The only difference from Example 1 is that in step 2, the incubation time is 60 minutes. The preparation method of the remaining single-cell nanocoatings is basically the same as that of Example 1.

[0099] Example 10: The only difference from Example 1 is that in the raw material preparation, the reagent containing the modifying material is DSPE-PSar2000-Maleimide, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSPE-PSar2000-Maleimide; in the buffer solution for preparing the cell suspension in the raw material preparation, Tris buffer is used instead of HEPES buffer. The preparation methods for the remaining single-cell nanocoatings are basically the same as in Example 1.

[0100] Example 11: The only difference from Example 1 is that in the raw material preparation, the reagent containing the modifying material is DSPE-PSar2000-Biotin, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSPE-PSar2000-Biotin. In the raw material preparation, PBS buffer is used instead of HEPES buffer in the buffer solution for preparing the cell suspension. The preparation methods for the remaining single-cell nanocoatings are basically the same as in Example 1.

[0101] Example 12: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is DSPE-PSar2000-Biotin, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSPE-PSar2000-Biotin. The preparation methods of the remaining single-cell nanocoatings are basically the same as those in Example 1.

[0102] Example 13: The only difference from Example 1 is that in the raw material preparation, the reagent containing the modifying material is prepared by weighing DSPE-PSar5000-NHS, dissolving it in an appropriate amount of DMSO, and preparing a reagent with a concentration of 20 mg / mL containing the modifying material. That is, in step 1, the modifying material is DSPE-PSar5000-NHS. In step 3, no stop agent is added, and the cells are directly washed twice with 10 mL of PBS solution, each time for about 5 minutes, to remove unreacted modifying material. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0103] Example 14: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is DSPE-PSar1000-NHS, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSPE-PSar1000-NHS; and in step 3, the terminating agent is 100 mM ethanolamine. The preparation methods of the remaining single-cell nanocoatings are basically the same as those in Example 1.

[0104] Example 15: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is DSPC-PSar2000-NHS, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSPC-PSar2000-NHS; in step 3, the terminating agent is 100 mM hydroxylamine. The preparation methods of the remaining single-cell nanocoatings are basically the same as those in Example 1.

[0105] Example 16: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is DSG-PSar2000-NHS ester, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSG-PSar2000-NHS ester. The preparation methods of the remaining single-cell nanocoatings are basically the same as in Example 1.

[0106] Example 17: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is Cholesterol-PSar2000-PFP ester, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is Cholesterol-PSar2000-PFP ester. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0107] Example 18: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is Dioctadecyl-PSar2000-DBCO, dissolved in an appropriate amount of DMSO, to prepare a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is Dioctadecyl-PSar2000-DBCO. The preparation methods for the remaining single-cell nanocoatings are basically the same as in Example 1.

[0108] Example 19: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is to weigh out Tocopherol-PSar2000-Thiol-ene, dissolve it in an appropriate amount of DMSO, and prepare a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is Tocopherol-PSar2000-Thiol-ene. The preparation method of the remaining single-cell nanocoatings is basically the same as in Example 1.

[0109] Example 20: The only difference from Example 1 is that the reagent containing the modifying material in the raw material preparation is DSPE-PSar2000, which is weighed, dissolved in an appropriate amount of DMSO, and prepared into a reagent containing the modifying material with a concentration of 20 mg / mL. That is, in step 1, the modifying material is DSPE-PSar2000. The preparation methods of the remaining single-cell nanocoatings are basically the same as those in Example 1.

[0110] Comparative Example 1: Hydrogel encapsulation scheme, the specific steps are as follows: 1. Preparation of hydrogel precursor solution: 10% w / v gelatin + 0.5 U / mL microbial transglutaminase (mTG), dissolved in PBS; 2. Cell embedding process: Cell suspension (1×10⁻⁶) is embedded in the cell membrane. 8 The cell / mL enzyme was mixed with the precursor solution at a ratio of 1:1 (v / v); the enzyme was cross-linked at 37°C to form hydrogel microspheres (200-500 μm in diameter).

[0111] 3. Post-processing: Wash with PBS to remove uncrosslinked precursors; equilibrate in a 37°C, 5% CO2 incubator for 2 h before use.

[0112] Comparative Example 2: The LBL (Layer-by-Layer) self-assembly scheme is adopted, and the specific steps are as follows: 1. Preparation of polyelectrolyte solution: Polycationic: Chitosan (CS, degree of deacetylation >85%) was dissolved in 10 mM HEPES buffer (pH 7.4); Polyanionic: 0.5 mg / mL sodium alginate (ALG), dissolved in the same buffer solution as above; 2. LBL self-assembly process: Step 1: Cell suspension (1×10⁻⁶) 8 (cells / mL) was mixed with a polycationic solution at a 1:1 ratio and incubated at 4°C for 10 min to form the first layer by electrostatic adsorption; Step 2: Wash twice with PBS to remove unadsorbed polycations; Step 3: Add polyanionic solution and incubate at 4°C for 10 min to form a second layer through electrostatic adsorption; Step 4: Repeat steps 2-3 to build multiple layers; Step 5: Select polyanionic (negative charge, anti-adhesion) material for the final layer.

[0113] 3. Post-processing: Wash with PBS to remove free polyelectrolytes; use after equilibration at 4°C or 37°C.

[0114] Test example: In the preparation process of Examples 1-20 and Comparative Examples 1-2, fluorescent markers were added when reagents containing modifying materials were added to facilitate subsequent fluorescence imaging and verification of the modification effect.

[0115] The single-cell nanocoatings obtained in Examples 1-20 and Comparative Examples 1-2 were subjected to the following tests.

[0116] I. Fluorescence Microscopy Test: The surface modification material containing the inserted fluorescent dye (FITC / CY5.5) and the modification material were mixed at a ratio of 1:100 as the actual modification material. Following the specific operating procedures of Examples 1-20, a portion of the cell suspension was aspirated and observed under a fluorescence microscope. The corresponding fluorescence module was selected and adjusted. A clear and continuous fluorescent ring was observed at the edge of the cell membrane. The test results of Examples 1-20 are shown below. Figures 2-6 .

[0117] Add the insertion fluorescent dye FITC to the hydrogel precursor solution. Follow the specific steps in Comparative Example 1, aspirate a portion of the cell suspension, and observe it under a fluorescence microscope. Select the corresponding fluorescence module and adjust it. You can observe an irregularly shaped, varying-thickness encapsulation layer outside the cells. The test results of Comparative Example 1 are shown below. Figure 7 .

[0118] Add the fluorescent dye FITC to the polyelectrolyte solution. Follow the specific steps in Comparative Example 2, aspirate a portion of the cell suspension, and observe it under a fluorescence microscope. Select the corresponding fluorescence module and adjust it. It can be observed that the edges of the cell inclusions exhibit irregular depressions and protrusions, and that there are significant differences in cell size between cells. The test results of Comparative Example 2 are shown below. Figure 8 .

[0119] II. Cell Viability Assay: The rejection assay was used. Based on the selective barrier function of intact cell membranes, 20 μL of cell suspension was mixed with an equal volume of dye (trypan blue / propidium iodide PI). 10 μL of the mixed suspension was added to a cell counting chamber. The cell counting chamber was inserted into an automated cell technology instrument or placed under a microscope for manual counting three times. The results showed that viable cells had intact cell membranes with selective permeability, rejecting the specific dye and preventing it from entering the cell, resulting in transparent / colorless cells. Dead cells lost cell membrane integrity, increased permeability, and the dye freely entered the cell and bound to nucleic acids, resulting in blue (trypan blue) or red fluorescence (PI). The test results for Examples 1-20 and Comparative Examples 1-2 are shown in Table 1.

[0120] Table 1

[0121] III. Single-cell nano-coating thickness test: The thickness of the single-cell nanocoating in Example 1 was tested using the acceptor photobleaching method. The specific steps included: Third-generation human adipose-derived mesenchymal stem cells were resuspended in DPBS and the concentration was adjusted to 1×10⁻⁶. 8 Cells / mL, add fluorescent donor DiI to a final concentration of 10 μg / mL, label at 37℃ in the dark with shaking for 20-30 min, centrifuge and wash to remove free dye. Following the method of this invention, anchor and modify with 50 μg / mL DSPE-PEG2000-FITC at 4℃ for 30 min, quench with 100 mM glycine for 5 min, wash twice with PBS, fix with 4% paraformaldehyde for 15 min, stain with DAPI nuclear dye, and mount with anti-quenching mounting medium.

[0122] A Zeiss LSM 880 laser scanning confocal microscope (63× oil immersion, NA 1.4) was used. DiI cells were excited with a 561 nm laser (2% power), and emission was detected at 570–620 nm. FITC cells were excited with a 488 nm laser (2% power), and emission was detected at 500–550 nm. DiI and FITC double-positive cells were selected, and the DiI fluorescence intensity within the region of interest (ROI) was recorded as I_DA = 1280 au (after background subtraction).

[0123] Subsequently, the ROI was irradiated with a 488 nm laser at 100% power for 45 s, and the FITC fluorescence was monitored in real time until it decreased by 96%. 10 s after bleaching was stopped, the DiI fluorescence intensity I_D = 2560 au was recorded with the same parameters (561 nm, 2% power).

[0124] The FRET efficiency is calculated as E = 1 - 1280 / 2560 = 0.50. Based on the Förster radius R0 of the DiI-FITC pair (5.5 nm), the coating thickness is calculated as r = 5.5 × [(1-0.5) / 0.5]^(1 / 6) = 5.5 × 1.0 = 5.5 nm. This conforms to the 1-10 nm thickness range described in this invention, constituting a nanoscale coating.

[0125] Results analysis: The test results above show that the cells modified with the single-cell nanocoatings in Examples 1-20 exhibited uniform fluorescence signals under a fluorescence microscope, indicating that the cell surface modification was uniform and successful. Furthermore, cell viability assays showed that the modified cells possessed good activity and intact cell membranes, thus maintaining good proliferation capacity and function in subsequent cultures.

[0126] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a single-cell nanocoating, characterized in that, The preparation method includes: S1. Add a reagent containing a modifying material to a cell suspension to obtain a mixture, wherein the modifying material includes a functionalized polysarcosine derivative; S2. Incubate the mixture to allow the cells and the modified material to react and form the single-cell nanocoating.

2. The preparation method according to claim 1, characterized in that, The structure of the functionalized polysarcosine derivative includes a front-end-intermediate polymer, wherein the front end is independently selected from DSPE, DOPE, DMPE, DPPE, DEPE, SOPE, DSG, Dialkylglycerol, POPE, Cholesterol, Dioctadecyl, Tocopherol, or Stearyl, and the intermediate polymer includes PSar.

3. The preparation method according to claim 2, characterized in that, The functionalized polysarcosine derivative further includes a posterior activating group. The structure of the functionalized polysarcosine derivative is a front-end-intermediate polymer-posterior activating group, wherein the posterior activating group is independently selected from NHS ester, Sulfo-NHS ester, NHS carbonate, PFPester, DBCO, Tetrazine, Norbornene, Cyclooctyne, TCO, Azide, Maleimide, Thiol-ene, Vinyl sulfone, Pyridyl disulfide, or Biotin.

4. The preparation method according to claim 1, characterized in that, The functionalized polysarcosine derivative is selected from DSPE-PSar-NHS ester, DSPE-PSar-Sulfo-NHS ester, DSPE-PSar-NHS carbonate, DSPE-PSar-PFP ester, DSPE-PSar-DBCO, DOPE-PSar-NHS ester, DOPE-PSar-Sulfo-NHS ester, DOPE-PSar-NHS carbonate, DOPE-PSar-PFP ester, DOPE-PSar-DBCO, DMPE-PSar-NHS ester, DMPE-PSar-Sulfo-NHS ester, DMPE-PSar-NHS carbonate, DMPE-PSar-PFP ester, DMPE-PSar-DBCO, DPPE-PSar-NHS ester, DPPE-PSar-Sulfo-NHS ester, DPPE-PSar-NHS carbonate, DPPE-PSar-PFP ester, DMPE-PSar-DBCO, DPPE-PSar-NHS ester, DPPE-PSar-NHS carbonate, DPPE-PSar-PFP ester, DPPE-PSar-PFP ester, DPPE-PSar-DBCO, DEPE-PSar-NHS ester, DEPE-PSar-Sulfo-NHS ester, DEPE-PSar-NHS carbonate, DEPE-PSar-PFP ester, DEPE-PSar-DBCO, SOPE-PSar-NHS ester, SOPE-PSar-Sulfo-NHS ester, SOPE-PSar-NHS carbonate, SOPE-PSar-PFP ester, SOPE-PSar-DBCO, DSG-PSar-NHS ester, DSG-PSar-Sulfo-NHS ester, DSG-PSar-NHS carbonate, DSG-PSar-PFP ester, DSG-PSar-DBCO, Dialkylglycerol-PSar-NHS ester, Dialkylglycerol-PSar-Sulfo-NHS ester, Dialkylglycerol-PSar-NHScarbonate, Dialkylglycerol-PSar-PFP ester, Dialkylglycerol-PSar-DBCO, POPE-PSar-NHS ester, POPE-PSar-Sulfo-NHS ester, POPE-PSar-NHScarbonate, POPE-PSar-PFPester, POPE-PSar-DBCO, Cholesterol-PSar-NHS ester, Cholesterol-PSar-Sulfo-NHSester, Cholesterol-PSar-NHS carbonate, Cholesterol-PSar-PFP ester, Cholesterol-PSar-DBCO, Dioctadecyl-PSar-NHS ester, Dioctadecyl-PSar-Sulfo-NHS ester, Dioctadecyl-PSar-NHS carbonate, Dioctadecyl-PSar-PFP ester, Dioctadecyl-PSar-DBCO, Tocopherol-PSar-NHS ester, Tocopherol-PSar-Sulfo-NHS ester, Tocopherol-PSar-NHS carbonate, Tocopherol-PSar-PFP ester、Tocopherol-PSar-DBCO、DSPE-PSar-Tetrazine、DSPE-PSar-Norbornene、DSPE-PSar-Cyclooctyne、DSPE-PSar-TCO、DSPE-PSar-Azide、DSPE-PSar-Maleimide、DSPE-PSar-Thiol-ene、DSPE-PSar-Vinyl sulfone、DSPE-PSar-Pyridyl disulfide、DSPE-PSar、DOPE-PSar-Tetrazine、DOPE-PSar-Norbornene、DOPE-PSar-Cyclooctyne、DOPE-PSar-TCO、DOPE-PSar-Azide、DOPE-PSar-Maleimide、DOPE-PSar-Thiol-ene、DOPE-PSar-Vinyl sulfone、DOPE-PSar-Pyridyldisulfide, DOPE-PSar, DMPE-PSar-Tetrazine, DMPE-PSar-Norbornene, DMPE-PSar-Cyclooctyne, DMPE-PSar-TCO, DMPE-PSar-Azide, DMPE-PSar-Maleimide, DMPE-PSar-Thiol-ene, DMPE-PSar-Vinylsulfone, DMPE-PSar-Pyridyl disulfide, DMPE-PSar, DPPE-PSar-Tetrazine, DPPE-PSar-Norbornene, DPPE-PSar-Cyclooctyne, DPPE-PSar-TCO, DPPE-PSar-Azide, DPPE-PSar-Maleimide, DPPE-PSar-Thiol-ene, DPPE-PSar-Vinyl sulfone, DPPE-PSar-Pyridyldisulfide, DPPE-PSar, DEPE-PSar-Tetrazine, DEPE-PSar-Norbornene, DEPE-PSar-Cyclooctyne, DEPE-PSar-TCO, DEPE-PSar-Azide, DEPE-PSar-Maleimide, DEPE-PSar-Thiol-ene, DEPE-PSar-Vinyl sulfone, DEPE-PSar-Pyridyl disulfide, DEPE-PSar, SOPE-PSar-Tetrazine, SOPE-PSar-Norbornene, SOPE-PSar-Cyclooctyne, SOPE-PSar-TCO, SOPE-PSar-Azide, SOPE-PSar-Maleimide, SOPE-PSar-Thiol-ene, SOPE-PSar-Vinyl sulfone, SOPE-PSar-Pyridyl disulfide、SOPE-PSar、DSG-PSar-Tetrazine、DSG-PSar-Norbornene、DSG-PSar-Cyclooctyne、DSG-PSar-TCO、DSG-PSar-Azide、DSG-PSar-Maleimide、DSG-PSar-Thiol-ene、DSG-PSar-Vinyl sulfone、DSG-PSar-Pyridyldisulfide, DSG-PSar, Dialkylglycerol-PSar-Tetrazine, Dialkylglycerol-PSar-Norbornene, Dialkylglycerol-PSar-Cyclooctyne, Dialkylglycerol-PSar-TCO, Dialkylglycerol-PSar-Azide, Dialkylglycerol-PSar-Maleimide, Dialkylglycerol-PSar-Thiol-ene, Dialkylglycerol-PSar-Vinyl sulfone, Dialkylglycerol-PSar-Pyridyl disulfide, Dialkylglycerol-PSar, POPE-PSar-Tetrazine, POPE-PSar-Norbornene, POPE-PSar-Cyclooctyne, POPE-PSar-TCO, POPE-PSar-Azide, POPE-PSar-Maleimide, POPE-PSar-Thiol-ene, POPE-PSar-Vinyl sulfone, POPE-PSar-Pyridyl disulfide, POPE-PSar, Cholesterol-PSar-Tetrazine, Cholesterol-PSar-Norbornene, Cholesterol-PSar-Cyclooctyne, Cholesterol-PSar-TCO, Cholesterol-PSar-Azide, Cholesterol-PSar-Maleimide, Cholesterol-PSar-Thiol-ene, Cholesterol-PSar-Vinyl sulfone, Cholesterol-PSar-Pyridyl disulfide, Cholesterol-PSar, Dioctadecyl-PSar-Tetrazine, Dioctadecyl-PSar-Norbornene, Dioctadecyl-PSar-Cyclooctyne, Dioctadecyl-PSar-TCO, Dioctadecyl-PSar-Azide, Dioctadecyl-PSar-Maleimide, Dioctadecyl-PSar-Thiol-ene, Dioctadecyl-PSar-Vinylsulfone, Dioctadecyl-PSar-Pyridyl disulfide, Dioctadecyl-PSar, Tocopherol-PSar-Tetrazine, Tocopherol-PSar-Norbornene, Tocopherol-PSar-Cyclooctyne, Tocopherol-PSar-TCO, Tocopherol-PSar-Azide, Tocopherol-PSar-Maleimide, Tocopherol-PSar-Thiol-ene, Tocopherol-PSar-Vinyl sulfone, Tocopherol-PSar-Pyridyl disulfide, Tocopherol-PSar, Stearyl-PSar-NHS ester, Stearyl-PSar-Sulfo-NHS ester, Stearyl-PSar-NHS carbonate, Stearyl-PSar-PFP ester、Stearyl-PSar-DBCO、Stearyl-PSar-Tetrazine、Stearyl-PSar-Norbornene、Stearyl-PSar-Cyclooctyne、Stearyl-PSar-TCO、Stearyl-PSar-Azide、Stearyl-PSar-Maleimide、Stearyl-PSar-Thiol-ene、Stearyl-PSar-Vinyl sulfone、Stearyl-PSar-Pyridyl disulfide、Stearyl-PSar、DSPE-PSar-Biotin、 At least one of DOPE-PSar-Biotin, DMPE-PSar-Biotin, DPPE-PSar-Biotin, DEPE-PSar-Biotin, DEPE-PSar-Biotin, SOPE-PSar-Biotin, DSG-PSar-Biotin, Dialkylglycerol-PSar-Biotin, POPE-PSar-Biotin, Cholesterol-PSar-Biotin, Dioctadecyl-PSar-Biotin, Tocopherol-PSar-Biotin, Stearyl-PSar-Biotin.

5. The preparation method according to claims 2-4, characterized in that, The molecular weight of PSar in the functionalized polysarcosine derivative is 500 Da-5000 Da.

6. The preparation method according to claim 1, characterized in that, In step S1, the concentration of the modifying material in the mixture is 10µg / mL-200µg / mL.

7. The preparation method according to claim 1, characterized in that, The incubation temperature in step S2 is 0℃-37℃; The incubation time is 10 min to 60 min.

8. The preparation method according to claim 1, characterized in that, The cells are selected from at least one of mesenchymal stem cells, natural killer cells, T cells, pancreatic islet cells, kidney cells, cardiomyocytes, liver cells, neural stem cells, dopaminergic progenitor cells, pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells.

9. The preparation method according to claim 1, characterized in that, The method for preparing the cell suspension includes: The cells were washed and the cell concentration was adjusted to 5 × 10⁻⁶ using a buffer solution. 5 cells / 300µL - 5×10 6 The cell suspension was obtained by dividing the cells by 300µL.

10. The preparation method according to claim 1, characterized in that, After step S2, the preparation method further includes: The cells in the mixture are washed by centrifugation.

11. The preparation method according to claim 10, characterized in that, Prior to the step of centrifuging and washing the cells in the mixture, the preparation method further includes: A terminator is added to the mixture to terminate the reaction, wherein the terminator is selected from at least one of glycine, ethanolamine, hydroxylamine, and DPBS.

12. A single-cell nanocoating, characterized in that, The single-cell nanocoating is prepared by the preparation method according to any one of claims 1-11.

13. A surface-engineered cell, characterized in that, The surface-engineered cells have the single-cell nanocoating as described in claim 12.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the surface-engineered cells of claim 13.

15. An engineered cell preparation, characterized in that, The engineered cell preparation includes the surface-engineered cells as described in claim 13.

16. A reagent kit, characterized in that, The kit comprises the surface-engineered cells of claim 13.

17. The use of the single-cell nanocoating of claim 12, the surface-engineered cell of claim 13, the pharmaceutical composition of claim 14, or the engineered cell preparation of claim 15 in cell labeling, drug delivery, and the preparation of medicaments for cell therapy.

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